We wish to create a technical infrastructure that would serve as a foundation for the study of pathophysiology of DNA CpG methylation in cancer. It has been shown that epigenetic modification of DNA in form of CpG methylation can result in mutations, cancer, and possibly aging. For example, a very clear mechanism has been established relating 5 MeC to an increase in mutational load through the transition of 5MeC to thymidine. In addition, changes in CpG methylation have been correlated with gene silencing, functional Loss Of Heterozygosity (LOH), and loss of epigenetic imprint. No current landscape allows for the study of changes in methylation at the single cell/single chromosome level. The central focus of this proposal is to establish a robust in situ technology for detection of sequence specific changes in CpG methylation on mammalian chromosomes. This technology would be sequence specific, capable of surveying multiple loci simultaneously, preserve the three dimensional organization of the metaphase chromosome, and be capable of ascertaining changes in cytosine methylation on metaphase chromosomes in single cells. To create this as a practical and transferable technology certain specific technical achievements can be defined. These incremental steps will be satisfied and the resulting technology evaluated in model systems to establish a base line of performance. This microscopic method will permit the investigator to distinguish the presence or absence of specific and/or global methylation in single cells at specific points in tumor development. This technology will also permit the investigator to develop an understanding of the normal development of the methylated genome in isolated single cells.